彩色马蹄莲细菌性软腐病菌的鉴定及其群体感应淬灭的研究
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摘要
彩色马蹄莲是近几年在国内外推出的中高档花卉,被公认为21世纪的“花卉之星”,但彩色马蹄莲细菌性软腐病发生严重,且没有理想的防治对策,极大阻碍了彩色马蹄莲在各地的发展。
本研究从不同品种的彩色马蹄莲病组织中分离到6个菌株,经形态特征与培养性状比较、生理生化测试、16S rDNA序列分析和致病性测定,结果表明6个分离物均为胡萝卜软腐果胶杆菌胡萝卜亚种(Pectobacterium carotovora subsp.carotovora)。此外,信号分子检测试验表明,分离菌株可产生并释放出N-酰基-高丝氨酸内酯(AHLs)类信号分子。
为了防治该病害,本研究利用信号分子干扰技术,通过群体感应淬灭酶(AttM解脂酶),降解病原菌的N-酰基-高丝氨酸内酯(AHLs)类信号分子,降低病原菌在马蹄莲上的致病性,从而达到防治该病害的目的。在研究中,我们一方面利用PCR技术扩增土壤根癌农杆菌EHA105中的attM基因,将其克隆到表达载体pUC19上,转化宿主菌DH5α,构建了DH5α/pUCM,用pUC19中的P_(lac)启动子表达attM基因;另一方面利用PCR技术,扩增大肠杆菌JM109中的P_(lpp)强启动子。随后通过重叠PCR,分别将P_(lpp)启动子与水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae)JXOⅢ中的hrfl基因和信号分子淬灭基因attM连接,连接于表达载体pUC19,构建工程菌JM109/pPHA,最终利用启动子P_(lpp)分别表达hrfl基因和attM基因。通过烟草过敏性反应,解酯酶AttM对病原菌信号分子降解作用的检测、以及attM基因编码的解酯酶AttM对彩色马蹄莲软腐病菌菌体生长和致病性的测定实验,表明:(1)自A.tumefaciens中获得的attM基因,在P_(lac)启动子下,在大肠杆菌中可以有效地表达并产生AttM(AHL降解酶),降解病原菌的信号分子;(2)在表达载体pUC19中,P_(lpp)启动子能同时有效表达attM基因和hrfl基因,可以引起烟草过敏性反应,同时降解病原菌的信号分子且对病原菌产生的信号分子的降解能力更强;(3)AttM解酯酶不影响彩色马蹄莲软腐病菌的菌体生长,但可以通过降解AHLs,影响软腐病菌的致病性,在活体和离体条件下可有效地减弱该病原菌的致病性,利用P_(lpp)启动子表达attM基因和hrfl基因,防治效果更加理想,不仅可以有效防治马蹄莲细菌性软腐病,还能促进植物本身生长,为有效防治彩色马蹄莲细菌性软腐病提供了一种新途径。
Recently,colored calla lily is becoming one of the high class flowers and described as "the star of flowers in 21 century".Due to the occurance of soft rot disease of colored calla lily and lack of effective ways to control this disease,the worldwide development of colored calla lily is seriously limited.
In this study,isolation and identification of the pathogen of soft rot of colored calla lily was performed.6 isolates were obtained from tissues of different colored calla lily.On the basis of morphological characterization,physiological and biochemical tests,16S rDNA sequence analysis and pathogenicty test,the 6 isolates were all identified as Pectobacterium carotovora subsp,carotovora.Additionally,the pathogen could produce N-acyl-homoserine lactone(AHL) molecular.
In order to control soft rot disease of colored calla lily,a biocontrol way was developed by quorum quenching technology using AHL-degrading enzyme ATTM.On one hand,the AHL-degrading enzyme gene attM was amplified from Agrobacterium tumefaciens strain EHA105 by PCR method and cloned into vector pUC19 for attM expression under promoter P_(lac).On the other hand,the strong and constitutive promoter P_(lpp) and hrf1 gene(encode harpin) were amplified from Escherichia coli strain JM109 and Xanthomonas oryzae pv.oryzae strain JXOⅢ,respectively,and spliced as P_(lpp)-hrf1 by overlap PCR method.Meanwhile,the promoter P_(lpp) and attM gene was also spliced as P_(lpp) -attM by overlap PCR method.Finally,the fragments P_(lpp)-hrf1 and P_(lpp)-attM were cloned into vector pUC19 and transformed into E.coli strain JM109,the positive clone was termed as JM109/P_(lpp)-hrf1-attM.Based on the results of hypersensitive response(HR), AHL-degrading bioassay and pathogenecity test,it was concluded that:(ⅰ) the attM gene was successfully expressed under the promoter P_(lac),and showed AHL degradation activity. (ⅱ) the hrf1 and attM genes were successfully expressed under strong promoter P_(lpp), respectively,in E.coli strian JM109,the clone JM109/P_(lpp)-hrf1-attM obtained the ability to induce HR in nonhost tobacco together with degrade AHL molecular.(ⅲ) the expressed AHL-degrading enzyme AttM could reduce the virulence of pathogen in in vivo and in vitro pathogenicity tests,but not affect the growth of pathogen.Moreover,the simultaneous expressed AttM and Harpin could not only reduce the virulence,but also promote the growth of color calla lily,and showed more effective to control this disease.Together,our results provided a new way for control of soft rot disease of color calla lily.
本研究从不同品种的彩色马蹄莲病组织中分离到6个菌株,经形态特征与培养性状比较、生理生化测试、16S rDNA序列分析和致病性测定,结果表明6个分离物均为胡萝卜软腐果胶杆菌胡萝卜亚种(Pectobacterium carotovora subsp.carotovora)。此外,信号分子检测试验表明,分离菌株可产生并释放出N-酰基-高丝氨酸内酯(AHLs)类信号分子。
为了防治该病害,本研究利用信号分子干扰技术,通过群体感应淬灭酶(AttM解脂酶),降解病原菌的N-酰基-高丝氨酸内酯(AHLs)类信号分子,降低病原菌在马蹄莲上的致病性,从而达到防治该病害的目的。在研究中,我们一方面利用PCR技术扩增土壤根癌农杆菌EHA105中的attM基因,将其克隆到表达载体pUC19上,转化宿主菌DH5α,构建了DH5α/pUCM,用pUC19中的P_(lac)启动子表达attM基因;另一方面利用PCR技术,扩增大肠杆菌JM109中的P_(lpp)强启动子。随后通过重叠PCR,分别将P_(lpp)启动子与水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae)JXOⅢ中的hrfl基因和信号分子淬灭基因attM连接,连接于表达载体pUC19,构建工程菌JM109/pPHA,最终利用启动子P_(lpp)分别表达hrfl基因和attM基因。通过烟草过敏性反应,解酯酶AttM对病原菌信号分子降解作用的检测、以及attM基因编码的解酯酶AttM对彩色马蹄莲软腐病菌菌体生长和致病性的测定实验,表明:(1)自A.tumefaciens中获得的attM基因,在P_(lac)启动子下,在大肠杆菌中可以有效地表达并产生AttM(AHL降解酶),降解病原菌的信号分子;(2)在表达载体pUC19中,P_(lpp)启动子能同时有效表达attM基因和hrfl基因,可以引起烟草过敏性反应,同时降解病原菌的信号分子且对病原菌产生的信号分子的降解能力更强;(3)AttM解酯酶不影响彩色马蹄莲软腐病菌的菌体生长,但可以通过降解AHLs,影响软腐病菌的致病性,在活体和离体条件下可有效地减弱该病原菌的致病性,利用P_(lpp)启动子表达attM基因和hrfl基因,防治效果更加理想,不仅可以有效防治马蹄莲细菌性软腐病,还能促进植物本身生长,为有效防治彩色马蹄莲细菌性软腐病提供了一种新途径。
Recently,colored calla lily is becoming one of the high class flowers and described as "the star of flowers in 21 century".Due to the occurance of soft rot disease of colored calla lily and lack of effective ways to control this disease,the worldwide development of colored calla lily is seriously limited.
In this study,isolation and identification of the pathogen of soft rot of colored calla lily was performed.6 isolates were obtained from tissues of different colored calla lily.On the basis of morphological characterization,physiological and biochemical tests,16S rDNA sequence analysis and pathogenicty test,the 6 isolates were all identified as Pectobacterium carotovora subsp,carotovora.Additionally,the pathogen could produce N-acyl-homoserine lactone(AHL) molecular.
In order to control soft rot disease of colored calla lily,a biocontrol way was developed by quorum quenching technology using AHL-degrading enzyme ATTM.On one hand,the AHL-degrading enzyme gene attM was amplified from Agrobacterium tumefaciens strain EHA105 by PCR method and cloned into vector pUC19 for attM expression under promoter P_(lac).On the other hand,the strong and constitutive promoter P_(lpp) and hrf1 gene(encode harpin) were amplified from Escherichia coli strain JM109 and Xanthomonas oryzae pv.oryzae strain JXOⅢ,respectively,and spliced as P_(lpp)-hrf1 by overlap PCR method.Meanwhile,the promoter P_(lpp) and attM gene was also spliced as P_(lpp) -attM by overlap PCR method.Finally,the fragments P_(lpp)-hrf1 and P_(lpp)-attM were cloned into vector pUC19 and transformed into E.coli strain JM109,the positive clone was termed as JM109/P_(lpp)-hrf1-attM.Based on the results of hypersensitive response(HR), AHL-degrading bioassay and pathogenecity test,it was concluded that:(ⅰ) the attM gene was successfully expressed under the promoter P_(lac),and showed AHL degradation activity. (ⅱ) the hrf1 and attM genes were successfully expressed under strong promoter P_(lpp), respectively,in E.coli strian JM109,the clone JM109/P_(lpp)-hrf1-attM obtained the ability to induce HR in nonhost tobacco together with degrade AHL molecular.(ⅲ) the expressed AHL-degrading enzyme AttM could reduce the virulence of pathogen in in vivo and in vitro pathogenicity tests,but not affect the growth of pathogen.Moreover,the simultaneous expressed AttM and Harpin could not only reduce the virulence,but also promote the growth of color calla lily,and showed more effective to control this disease.Together,our results provided a new way for control of soft rot disease of color calla lily.
引文
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